The Activating switch of Zn2+ by the mutation of the amino acid Tyrosine

to Alanine in the Dopamine Transporter

protein possibly impairing cocaine’s

effect on locomotion

Cocaine in the Nervous System

Stimulant

Science.education.nih.gov

Short term use Increased blood pressure Increased heart rateRisk of cardiac arrest

Long term use Changes the chemistry of the brain

What’s the significance of Zinc in Dopamine Transporter ?

Loland et al

Can this be done in C.elegans to further examine if the

switch of Zn2+ by the mutation of Tyrosine to alanine can possibly cocaine’s effect in

locomotion ?

Biology.syr.edu

They are the simplest organisms with a nervous system

They have their whole genome sequenced.

They are easily accessible.

Has short generation time.

Cost and time effective.

Which gene codes for DAT in C.elegans?

Dat-1 gene codes for C.elegans

Located on Chromosome III

Ncbi.nlm.nih.gov

Does the Same Tyrosine used in hDAT mutation exist in

C.elegans?

EMBOSSEbi.ac.uk

C.Elegans DAT

Human DAT

What mRNA codons are responsible for the

production of Tyrosine?

Start ORF program.

(Star.mit.eu)

Possible codons for Tyrosine:UACUAU

Biochem.com

UAC

UAUTyrosine

GCUGCCGCAGCC

Alanine

CRISPR/CAS9Method

Sander et al

Sander et al

Measure effectiveness of mutation by analyzing

cocaine’s ability to inhibit dopamine uptake in Y328A

DAT both in the presence and absence of Zn2+

Transfection Method

Niborlab.com

Ibioexperts.com

Possible Outcomes

Dopamine Uptake percent is going to be greater in the mutated DAT compared to wild type as concentration of cocaine increases.

Dopamine Uptake percent of the mutated DAT is going to be identical to wild type as concentration of cocaine increases.

Dopamine Uptake percent is less in the mutated DAT compared to wild type as concentration of cocaine increases.

Loland et alCocaine’s ability to inhibit DA uptake has been impaired

Loland et al

Cocaine’s ability to impair DA uptake has not been affected

Loland et alCocaine’s ability to impair DA uptake has been amplified.

Possible Outcomes

Dopamine Uptake percent is going to be greater in the mutated DAT compared to wild type as concentration of cocaine increases.

Dopamine Uptake percent of the mutated DAT is going to be identical to wild type as concentration of cocaine increases.

Dopamine Uptake percent is less in the mutated DAT compared to wild type as concentration of cocaine increases.

Further experiments to see whether or not mutated dopamine transport makes C.elegens less susceptible to behavioral effects of cocaine (i.e locomotion)

References Loland, C. J., Norregaard, L., Litman, T., & Gether, U. (2002). Generation of an activating Zn2+ switch in the dopamine transporter: Mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1683–1688. doi:10.1073

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122251/pdf/pq0302001683.pdf

Musselman, H. N., Neal-Beliveau, B., Nass, R., & Engleman, E. (2012). Chemosensory Cue Conditioning with Stimulants in a Caenorhabditis elegans Animal Model of Addiction. Behavioral Neuroscience, 126(3), 445–456. doi:10.1037

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367381/pdf/nihms368977.pdf

Nian-Hang Chen, Maarten E. A. Reith, Michael W. Quick. (2004). Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch - Eur J Physiol 447:519–531 DOI 10.1007/

http://link.springer.com/article/10.1007%2Fs00424-003-1064-5#

Gether, Ulrik, Andersen, Peter H., Larsson, Orla M, Schousboe, Arne. (2006). Neurotransmitter transporters: molecular function of importantdrug targets. TRENDS in Pharmacological Sciences Vol.27 No.7. 375-383

http://www.sciencedirect.com/science/article/pii/S0165614706001271

Norregaard, L., Frederiksen, D., Nielsen, E. O. & Gether, U. (1998) EMBO J. 17, 4266–4273.

Lo, T.-W., Pickle, C. S., Lin, S., Ralston, E. J., Gurling, M., Schartner, C. M., … Meyer, B. J. (2013). Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions. Genetics, 195(2), 331–348. doi:10.1534

http://www-ncbi-nlm-nih-gov.proxy.library.vcu.edu/pmc/articles/PMC3781963/

Chiu, H., Schwartz, H. T., Antoshechkin, I., & Sternberg, P. W. (2013). Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics, 195(3), 1167–1171. doi:10.1534

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813845/pdf/1167.pdf